Removal of glutathione produces apoptosis and necrosis in HepG2 cells overexpressing CYP2E1

Background: Previous studies have shown that addition of ethanol, iron, or arachidonic acid to HepG2 cells expressing CYP2E1 produced a loss in cell v iability and caused apoptosis. These effects were enhanced when cellular re duced glutathione (GSH) levels were lowered by treatment with buthionine su lfoximine (BSO). Overexpression of CYP2E1 in HepG2 cells could produce toxi city even in the absence of added toxin after BSO treatment. Studies were c arried out to characterize this CYP2E1-and BSO-dependent toxicity. Methods: HepG2 cells expressing CYP2E1 were treated with BSO for 1 to 4 day s, and various parameters associated with apoptosis and cell viability were assayed. Results: Treatment of cells expressing CYP2E1 (E47 cells) with BSO resulted in apoptosis as wen as necrosis. The apoptosis and necrosis were independe nt of each other. No toxicity was found with control HepG2 cells or HepG2 c ells expressing CYP3A4 instead of CYP2E1 under these conditions. The antiox idant trolox partially prevented the apoptosis and necrosis, whereas dially lsulfide, a CYP2E1 inhibitor, was fully protective. The activity of caspase 3, but not caspases 1, 8, or 9, was increased in the BSO-treated E47 cells , and an inhibitor of caspase 3 prevented apoptosis. Damage to mitochondria appears to play a role in the CYP2E1- and BSO-dependent toxicity, because mitochondrial membrane potential was decreased and cyclosporin A, an inhibi tor of the mitochondrial membrane permeability transition, prevented the ap optosis and the necrosis. The fall in membrane potential was prevented by t rolox and diallylsulfide, suggesting damage to the mitochondria by CYP2E1-d erived reactive oxygen species. Conclusions: These results indicate the critical role of GSH in protecting against CYP2E1-mediated oxidative stress and that mitochondria may be a tar get for CYP2E1-derived reactive oxygen species, and suggest that interactio ns between CYP2E1, mitochondria, and altered GSH homeostasis may play a rol e in alcohol-induced liver injury.